With marked advances in medical technology, it has recently become popular to perform organ transplants, i.e., replacing a difficult-to-treat organ with another person's organ. The organs that can be transplanted are quite diverse and include the skin, cornea, kidney, liver and heart, and in addition, the postoperative progress of organ transplants has improved so remarkably that they are already becoming established as a medical procedure. Keratoplasty is one example and as early as 40 years ago, an eye bank was organized in Japan to start transplanting activities. However, as of today, the number of donors in Japan is very small and notwithstanding the fact that there are annually about 20,000 patients who need keratoplasty, only a tenth of them (ca. 2,000 in number) can actually be treated by that procedure. Although keratoplasty is a virtually established procedure, it suffers the problem of shortage in donors, giving rise to the need for the development of a next-generation medical procedure.
With this background, attention has been drawn to the procedure of directly transplanting artificial substitutes or cells that were cultured into assembly. Typical examples of this approach are the artificial skin and the cultured skin. However, the artificial skin using synthetic polymers has the potential to cause rejection and other side effects that make it undesirable as skin grafts. On the other hand, the cultured skin is prepared by cultivating a portion of the normal skin of the patient until it grows to a desired size, so it can be used without the risk of causing rejection and any other side effects and may well be described as the most natural masking agent.
Conventionally, such cell culture has been performed either on the surface of glass or on the surface of synthetic polymers that were subjected to a variety of treatments. For example, a variety of polystyrene vessels that were subjected to surface treatments such as y-ray irradiation and silicone coating have become popular for use in cell culture. Cells that have been cultivated to grow on those vessels for cell culture are detached and recovered from the surfaces of the vessels by treatment with proteinases such as trypsin or chemical reagents.
However, it has been pointed out that the recovery of grown cells by treatment with chemical reagents involves some disadvantages such as the processing steps becoming cumbersome to increase the chance of contamination by impurities and the grown cells becoming denatured or damaged by the chemical treatment to have their inherent functions injured. In order to overcome these disadvantages, several techniques have been proposed to date.
JP 2-23191 B describes a method for producing a transplantable membrane of keratin tissue which comprises the steps of cultivating human neonatal keratinized epidermic cells in a culture vessel under conditions that enable a membrane of keratin tissue to form on the surface of the vessel and detaching the membrane of keratin tissue using an enzyme. Specifically, with 3T3 cells used as a feeder layer, the epidermic cells are grown and stratified as a cell sheet which is recovered using the proteinase dispase. However, the method described in JP 2-23191 B has had the following defects.    (1) Dispase is of microbial origin and the recovered cell sheet needs to be washed thoroughly.    (2) The conditions for dispase treatment differ from one batch of cell culture to another and great skill is required in the treatment.    (3) The cultured epidermic cells are pathologically activated by dispase treatment.    (4) The extracellular matrix is decomposed by dispase treatment.    (5) As the result, the diseased site to which the cell sheet has been grafted is prone to infection.
However, anterior ocular segment related cells that are contemplated in the present invention, such as corneal epithelial cells, corneal endothelial cells and conjunctival epithelial cells, do not have as strong intercellular binding as dermal cells and it has been impossible to detach and recover cultivated cells as a single sheet even if the dispase is employed.
In order to solve this problem, a technique has recently been devised, according to which corneal epithelial cells or conjunctival epithelial cells are cultured into assembly on an amnion deprived of the spongy layer and the epithelial layer and the assembly is used as a cell graft together with the amnion (JP 2001-161353 A). Since the amnion has adequate strength as a membrane but has no antigenicity, it is favorable as a support of cell grafts; however, the amnion is not inherently in the eye and in order to construct a more precise intraocular tissue, it has been desired that a satisfactorily strong sheet be prepared solely from the intraocular cells.
The present invention has been accomplished with a view to solving the aforementioned problems of the prior art. Therefore, the present invention has as an object providing an anterior ocular segment related cell sheet or a three-dimensional structure that have only a few structural defects as they have been recovered retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate. Another object of the present invention is to provide a process by which cultivated and grown cells can be detached and recovered from a substrate's surface easily and as a satisfactorily strong, single sheet by changing the ambient temperature without treatment with an enzyme such as dispase.